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US3676298A - Process for the treatment of organs to be used as grafts,notably animal or human corneas - Google Patents

Process for the treatment of organs to be used as grafts,notably animal or human corneas Download PDF

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US3676298A
US3676298A US42748A US3676298DA US3676298A US 3676298 A US3676298 A US 3676298A US 42748 A US42748 A US 42748A US 3676298D A US3676298D A US 3676298DA US 3676298 A US3676298 A US 3676298A
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corneas
grafts
treatment
cornea
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US42748A
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Madeleine Csanadi Moczar
Elemer Moczar
Paul Bertrand Payrau
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Bpifrance Financement SA
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts

Definitions

  • the invention relates to the treatment of corneas removed from human beings or animals and adapted to be subsequently grafted.
  • lproducts capable of establishing bridge-links between the protein molecules of the corneas are used to treat excised corneas to reduce the capacity of inflation of said corneas when they have been grafted while retaining their transparency.
  • These products are notably dialdehydes.
  • the invention applies more particularly to transfxion grafts for the surgical treat-ment of stula corneas, pterygium and especially ychronic edema of the cornea.
  • the invention relates to the treatment of animal or human organs with a view to their future use, notably as grafts, and it relates more particularly, though not exclusively, to a process for the treatment of corneas, as this is the case in which its application appears to be the most useful.
  • corneal grafts for the treatment of ocular diseases are divided into two main types: lamellar grafts or transxion grafts.
  • the rst of these techniques consists in operating on a relatively thin layer of the cornea, retaining the endothelial membrane intact on the organ treated. It is known that the endothelium plays a fundamental role in the control of corneal hydration, which conditions the transparency of the cornea.
  • This type of lamellar graft presupposes the existence of ⁇ a functionally intact endothelial layer on the organ treated.
  • IProcesses for preserv-ing corneas essentially for lamellar grafts are known in the prior art. These processes generally consist in maintaining corneas in a dried or frozen state after their removal. Such corneas can be used, as required .as lamellar grafts when -a normally functioning endothelial layer is present. Unfortunately, they cannot be used as transxion grafts because, after a few days, they swell and become rapidly opaque. This swelling and opacity are due to the natural tendency of the cornea to ment with a malachite green solution and an antibiotic physiological solution before the removal of the cornea. The cornea is also washed in an antibiotic physiologic solution and is then secured to a cylindrical support. Desiccation is effected by means of ⁇ an aluminium gel.
  • An object of the invention is a process for treating corneas with a View to their subsequent use as transfixion or lamellar grafts, a process which does not have the abovementioned drawbacks.
  • this process consists essentially in causing products capable of establishing bridge-links 'between the protein molecules of the cornea to act on the said cornea.
  • the single drawing shows an example of an embodiment of a device for holding corneas -treated according to the' process of the invention during the de-siccation step.
  • the process of treatment according to the invention can be applied to corneas surrounded with the sclera or in isolation, or corneas without the epithelium and endothelium (stroma).
  • dialdehydes which are capable of establishing bridge-links between the protein molecules of corneas are used to treat corneas.
  • Corneas can be treated with C2 to C6 dialdehydes, and preferably with glyoxal and glutaraldehyde.
  • the corneas are immersed in an aqueous aldehyde solution having a concentration in the range of 0.5 to 20%.
  • the time required for treatment may vary from 30 minutes to 2 hours in the case of glutaraldehyde and from 1 to 3 hours for glyoxal.
  • the corneas are exposed to vapors of dialdehydes for a period of time varying from about l2 to 60 hours.
  • dialdehyde wherein the two aldehyde groups are separated by 1 to 4 carbon atoms, it being possible to replace one of these atoms by an oxygen atom.
  • the chain separating the carbonyls can be branched and substituted with one -or more OH groups.
  • dialdehydes can be formed in situ by oxidizing the stroma carbohydrates with the salts of a periodic acid.
  • the corneas are soaked in a buffered or unbulfered solution of NaIO4, for instance, at a temperature in the range of from 4 to 25 C. and in the dark.
  • the treatment can last from 1 to 24 hours.
  • the sodium periodate is destroyed by a glycol solution at a concentration of 0.1 to 2%.
  • the gl-ucide chains of the stroma chains are thus converted into polyaldehydes by cleavage of the bonds
  • the excess reagent is eliminated by washing the cornea in distilled water after the cornea has been treated with the dialdehyde or periodate.
  • the cornea is desiccated on a silica gel or on calcium chloride at room temperature and for a period of about two days. Intensive desiccation should be avoided as it may destroy the histologic structure of the cornea.
  • the desiccation process is an essential element in the formation of bridge-links. For this reason, it is preferable to preseve the corneas so treated in the dry, rather than in the frozen state.
  • the desiocation step is preferably carried out by maintaining the cornea in its natural shape by means of a suitable device.
  • a suitable device comprising a
  • corneas When corneas are used, they are subjected to preliminary washing for several hours in a physiological salt solution to remove traces of the reagent.
  • EXAMPLE 1 The corneal epithelium is removed from human eyeballs within 24 hours after death. The cornea is then incised and removed, 8 to 10 mm. of the solera being retained. It is cleaned with a scarilicator and rinsed in physiological salt solution. It is then soaked in 30 ml. of a 3% glyoxal solution for 1 hour at 4 C., with occasional stirring. The cornea with its surrounding sclera is then washed several times in physiological salt solution, with decantation each time. ⁇ It is then placed on the device shown in the gure to be desiccated for 48 hours by means of a dehydrated silica gel at 120 C.
  • Corneas so treated are then soaked several times in physiological salt solution for one hour, with decantation each time, they are then dried in a desiccator by means of dehydrated silica, but without placing them on the device of the figure.
  • the corneas can then be preserved for several months.
  • the corneas are then rehydrated when they are required for use. Their maximum level of rehydration is from '0.7 to 0.8 of the normal physiological level. These corneas are perfectly transparent.
  • EXAMPLE 2 Excised pig corneas are immersed for 5 hours in a 1% aqueous solution of NaIO4 in darkness and at a temperature of 4 C. The corneas are then rinsed in Water and soaked for 2 hours in a 0.2% aqueous glycol solution. The Icorneas are then Washed and desiccated according to the process given in Example 1. The hydration level of corneas so treated is from 0.6 to 0.7 of the normal physiological hydration level.
  • the hydration level of these corneas is from 0.6 to 0.8 that of the normal physiological level.
  • the process of the invention enables corneas to be obtained which are useable as transxion grafts despite the absence of a living endothelium.
  • the grafts obtained by this process have a hydration level compatible with transparence.
  • the invention can be applied to the surgical treatment of fistula corneas, pterygium and especially chronic edemas of the cornea.
  • a process for treating corneas to prevent their swelling and losing their transparency for future use as grafts comprising establishing bridge-links between the protein molecules of the constituent substances of said corneas and dialdehydes by 1) placing said corneas in intimate contact with a bridge linking reagent selected from the group consisting of a dialdehyde having 2 to 6 carbon atoms in the form of an aqueous solution containing 0.5-20% of said dialdehyde, a vapor or formed in situ with a 1% aqueous solution of NaI-O4 without effecting the transparency of the cornea; (2) Washing the corneas to remove the excess reagent from the corneas; and (3) partially desiccating the corneas to facilitate the formation of said bridge links without destroying the histologic structure of the cornea.
  • a bridge linking reagent selected from the group consisting of a dialdehyde having 2 to 6 carbon atoms in the form of an aqueous solution containing 0.5-20% of said dialdehyde, a vapor or formed in situ with
  • dialdehydes are formed in situ on the corneas by the oxydation of the carbohydrates of the stroma by soaking in a 1% Nal04 solution at a temperature of 4-25 C., in the dark for l to 24 hours.
  • dialdehyde is in the form of a 0.5 to 20% aqueous solution.
  • dialdehyde is glutaraldehyde and said contact time is 30 minutes to 2 hours.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
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Abstract

THE INVENTION RELATES TO THE TREATMENT OF CORNEAS REMOVED FROM HUMAN BEINGS OR ANIMALS AND ADAPTED TO BE SUBSEQUENTLY GRAFTED. ACCORDING TO THE PROCESS OF THE INVENTION, PRODUCTS CAPABLE OF ESTABLISHING BRIDGE-LINKS BETWEEN THE PROTEIN MOLECULES OF THE CORNEAS ARE USED TO TREAT EXCISED CORNEAS TO REDUCE THE CAPACITY OF INFLATION OF SAID CORNEAS WHEN THEY HAVE BEEN GRAFTED WHIEL RETAINING THEIR TRANSPARENCY. THESE PRODUCTS ARE NOTABLY DIALDEHYDES. THE INVENTION APPLIES MORE PARTICULARLY TO TRANSFIXION GRAFTS FOR THE SURGICAL TREATMENT OF FISTULA CORNEAS, PTERYGIUM AND ESPECIALLY CHRONIC EDEMA OF THE CORNEA.

Description

July l1, 1972 M. c. MoczAR ETAL 3,676,298
PROCESS FOR THE TREATMENT OF ORGANS To BE USED AS GRAFTS, NOTABLY ANIMAL OR HUMAN CORNEAS Filed June 2, 1970 In welfare United States Patent Olce 3,676,298 Patented July l1, 1972 PROCESS FOR THE TREATMENT F ORGANS T0 BE USED AS GRAFTS, NOTABLY ANIMAL 0R HUMAN CORNEAS Madeleine Csanadi Moczar and Elemer Moczar, Gif-sur- Yvette, and Paul Bertrand Payrau, Paris, France, assignors to Etablissement Public: Agence Nationale de Valorisation de la Recherche (ANVAR), Courbevoie,
France Filed June 20, 1970, Ser. No. 42,748 Claims priority, application France, June 3, 1969, 6 18127 Int. Cl. A61f 1/16, 9/00 U.S. Cl. 195-1.7 9 Claims ABSTRACT OF THE DISCLOSURE The invention relates to the treatment of corneas removed from human beings or animals and adapted to be subsequently grafted.
According to the process of the invention, lproducts capable of establishing bridge-links between the protein molecules of the corneas are used to treat excised corneas to reduce the capacity of inflation of said corneas when they have been grafted while retaining their transparency. These products are notably dialdehydes.
The invention applies more particularly to transfxion grafts for the surgical treat-ment of stula corneas, pterygium and especially ychronic edema of the cornea.
The invention relates to the treatment of animal or human organs with a view to their future use, notably as grafts, and it relates more particularly, though not exclusively, to a process for the treatment of corneas, as this is the case in which its application appears to be the most useful.
It is known that corneal grafts for the treatment of ocular diseases are divided into two main types: lamellar grafts or transxion grafts. i The rst of these techniques consists in operating on a relatively thin layer of the cornea, retaining the endothelial membrane intact on the organ treated. It is known that the endothelium plays a fundamental role in the control of corneal hydration, which conditions the transparency of the cornea. This type of lamellar graft presupposes the existence of `a functionally intact endothelial layer on the organ treated.
It is necessary to use the second technique when the endothelial layer of the organ treated is also affected, as is the case, for instance, in chronic edmea of the cornea. It consists in operating on the cornea and endothelium as a whole.
IProcesses for preserv-ing corneas essentially for lamellar grafts are known in the prior art. These processes generally consist in maintaining corneas in a dried or frozen state after their removal. Such corneas can be used, as required .as lamellar grafts when -a normally functioning endothelial layer is present. Unfortunately, they cannot be used as transxion grafts because, after a few days, they swell and become rapidly opaque. This swelling and opacity are due to the natural tendency of the cornea to ment with a malachite green solution and an antibiotic physiological solution before the removal of the cornea. The cornea is also washed in an antibiotic physiologic solution and is then secured to a cylindrical support. Desiccation is effected by means of `an aluminium gel.
Other known processes aim at maintaining the graft in survival by means of an isolated organ culture method. U.S.A. Pat. No. 3,371,012 for instance, describes a medium which ensures the necessary physiological conditions for cells to remain viable for several days. This medium is a solution containing a mucopolysaccharide acid. Similarly, French Pat. No. 1,472,970 relates to the preservation of corneal grafts by means of a liquid containing a mucopolysaccharide acid and vitamin C. However, the cornea is a living tissue whose preservation depends on the viability of the cells, and consequently cannot be preserved by these processes for longer than about seven days.
An object of the invention is a process for treating corneas with a View to their subsequent use as transfixion or lamellar grafts, a process which does not have the abovementioned drawbacks.
Broadly speaking, this process consists essentially in causing products capable of establishing bridge-links 'between the protein molecules of the cornea to act on the said cornea.
Other characteristics and advantages of the invention will be brought out from the description which follows and from the appended drawing.
The single drawing shows an example of an embodiment of a device for holding corneas -treated according to the' process of the invention during the de-siccation step.
Examples of the embodiment of the process of the invention will be given hereinafter, it being understood that these examples are in no Way limitative.
The process of treatment according to the invention can be applied to corneas surrounded with the sclera or in isolation, or corneas without the epithelium and endothelium (stroma).
According to the invention, dialdehydes which are capable of establishing bridge-links between the protein molecules of corneas are used to treat corneas.
It has notably been discovered that certain dialdehydes easily form bridge-links with the proteins of corneal stroma, with the result that their hydration capacity is decreased.
Corneas can be treated with C2 to C6 dialdehydes, and preferably with glyoxal and glutaraldehyde.
According to a first, preferred, embodiment of the invention, the corneas are immersed in an aqueous aldehyde solution having a concentration in the range of 0.5 to 20%. The time required for treatment may vary from 30 minutes to 2 hours in the case of glutaraldehyde and from 1 to 3 hours for glyoxal.
According to another mode of embodiment of the process of the invention, the corneas are exposed to vapors of dialdehydes for a period of time varying from about l2 to 60 hours.
It is also possible to use a dialdehyde wherein the two aldehyde groups are separated by 1 to 4 carbon atoms, it being possible to replace one of these atoms by an oxygen atom. The chain separating the carbonyls can be branched and substituted with one -or more OH groups.
According to another preferred from of embodiment of the process of the invention, dialdehydes can be formed in situ by oxidizing the stroma carbohydrates with the salts of a periodic acid. The corneas are soaked in a buffered or unbulfered solution of NaIO4, for instance, at a temperature in the range of from 4 to 25 C. and in the dark. The treatment can last from 1 to 24 hours.
The sodium periodate is destroyed by a glycol solution at a concentration of 0.1 to 2%. The gl-ucide chains of the stroma chains are thus converted into polyaldehydes by cleavage of the bonds In the embodiments given hereinabove, the excess reagent is eliminated by washing the cornea in distilled water after the cornea has been treated with the dialdehyde or periodate. The cornea is desiccated on a silica gel or on calcium chloride at room temperature and for a period of about two days. Intensive desiccation should be avoided as it may destroy the histologic structure of the cornea.
It should be noted that the desiccation process is an essential element in the formation of bridge-links. For this reason, it is preferable to preseve the corneas so treated in the dry, rather than in the frozen state.
The desiocation step is preferably carried out by maintaining the cornea in its natural shape by means of a suitable device. The appended figure shows an example of an embodiment of such a device. It comprises a |base onto which is secured a piece of revolution 2, the upper portion 3 of which has the shape and size of an eye. 4 represents the cornea surrounded with its sclera 5 stretched over the form 3 by means of suture threads such as 6 and 7. These threads are attached to xing hooks such as 8 and 9 rigidly secured to base 1.
When corneas are used, they are subjected to preliminary washing for several hours in a physiological salt solution to remove traces of the reagent.
Some examples of the treatment of corneas according to the process of the invention, and the results obtained, will be given hereinbelow.
EXAMPLE 1 The corneal epithelium is removed from human eyeballs within 24 hours after death. The cornea is then incised and removed, 8 to 10 mm. of the solera being retained. It is cleaned with a scarilicator and rinsed in physiological salt solution. It is then soaked in 30 ml. of a 3% glyoxal solution for 1 hour at 4 C., with occasional stirring. The cornea with its surrounding sclera is then washed several times in physiological salt solution, with decantation each time. `It is then placed on the device shown in the gure to be desiccated for 48 hours by means of a dehydrated silica gel at 120 C.
Corneas so treated are then soaked several times in physiological salt solution for one hour, with decantation each time, they are then dried in a desiccator by means of dehydrated silica, but without placing them on the device of the figure.
The corneas can then be preserved for several months.
The corneas are then rehydrated when they are required for use. Their maximum level of rehydration is from '0.7 to 0.8 of the normal physiological level. These corneas are perfectly transparent.
Substantially similar results are obtained by replacing the glyoxal used in this example with glutaraldehyde.
EXAMPLE 2 Excised pig corneas are immersed for 5 hours in a 1% aqueous solution of NaIO4 in darkness and at a temperature of 4 C. The corneas are then rinsed in Water and soaked for 2 hours in a 0.2% aqueous glycol solution. The Icorneas are then Washed and desiccated according to the process given in Example 1. The hydration level of corneas so treated is from 0.6 to 0.7 of the normal physiological hydration level.
4 EXAMPLE 3 Moist pig corneas are exposed to glyoxal vapour in equilibrium with a 30% solution of this aldehyde for 48 hrs. in darkness and at a temperature of about 20 C.
These corneas are then desiccated according to the process described in Example 1. The hydration level of these corneas is from 0.6 to 0.8 that of the normal physiological level.
It is seen that the process of the invention enables corneas to be obtained which are useable as transxion grafts despite the absence of a living endothelium. The grafts obtained by this process have a hydration level compatible with transparence.
The invention can be applied to the surgical treatment of fistula corneas, pterygium and especially chronic edemas of the cornea.
What we claim is:
1. A process for treating corneas to prevent their swelling and losing their transparency for future use as grafts comprising establishing bridge-links between the protein molecules of the constituent substances of said corneas and dialdehydes by 1) placing said corneas in intimate contact with a bridge linking reagent selected from the group consisting of a dialdehyde having 2 to 6 carbon atoms in the form of an aqueous solution containing 0.5-20% of said dialdehyde, a vapor or formed in situ with a 1% aqueous solution of NaI-O4 without effecting the transparency of the cornea; (2) Washing the corneas to remove the excess reagent from the corneas; and (3) partially desiccating the corneas to facilitate the formation of said bridge links without destroying the histologic structure of the cornea.
2. The process of claim 1, in which said corneas are desiccated on a drying agent selected from the group consisting of silica gel and calcium chloride.
3. A process according to claim 1 wherein said corneas are exposed to a dialdehyde vapor reagent for a period of 12 to 60 hours.
4. A process according to claim 1 wherein the two aldehyde groups in said dialdehyde are separated by 1 to 4 carbon atoms.
5. A process according to claim 1 wherein said dialdehydes are formed in situ on the corneas by the oxydation of the carbohydrates of the stroma by soaking in a 1% Nal04 solution at a temperature of 4-25 C., in the dark for l to 24 hours.
6. The process of claim 1 in which the dialdehyde is in the form of a 0.5 to 20% aqueous solution.
7. A process according to claim 6 wherein said treatment is carried out for a period of time of from 0.5 to 48 hours at a temperature in the range of 0 to 37 C.
8. The process of claim 7 in which said dialdehyde is glutaraldehyde and said contact time is 30 minutes to 2 hours.
9. The process of claim 7 in which said dialdehyde is glyoxal, and said contact time is 1-3 hours.
References Cited UNITED STATES PATENTS 3,057,775 10/ 1962 Rendon 424-333 FOREIGN PATENTS 1,063,330 1/1960 Germany 195-1.7
OTHER REFERENCES Yanoff et al., Amer. J. of Clin. IPath., vol. 44, pp. 161-171, August 1965.
RICHARD L. HUFF, Primary Examiner U.S. Cl. XR. 424--3 33 .P0-V250 UNITED STATES` PATENT O FFCE 5/u) 4 b CERTIFICATE OF CORRECTION Paten-NO. '3,676,298 I I Dtedr- July 11. 1972 i Inventer) v MOCZr -et al s It is certified that error. appears in the above-identified patent and that said Letters Patent are hereby Corrected as. shown below:
Page l, Colufnn l, the filinq .date should read g Filed June 1970, ser. No. 42,748.
Signed and sealed this 12thl day -oflDecember' .1972.
(SEAL) Attest:
EDWARD M.FLRTCRRR,JR. ROBERT GOTTSCRALK Attesting Officer Commssionenof Patent
US42748A 1969-06-03 1970-06-20 Process for the treatment of organs to be used as grafts,notably animal or human corneas Expired - Lifetime US3676298A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4242291A (en) * 1979-05-09 1980-12-30 Battelle Development Corporation Collagen reconstitution
US4361552A (en) * 1980-09-26 1982-11-30 Board Of Regents, The University Of Texas System Wound dressing
US5077218A (en) * 1988-05-05 1991-12-31 Oregon Lions Sight And Hearing Foundation Inc. Eye transport apparatus
US5328821A (en) * 1991-12-12 1994-07-12 Robyn Fisher Cold and cryo-preservation methods for human tissue slices
US10969210B2 (en) 2018-04-25 2021-04-06 Pro-Tracker Ip Holding, Llc System and method for adjusting the trajectory of an arrow

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0627879A1 (en) * 1991-09-20 1994-12-14 CAMIENER, Gerald, W. Methods and compositions with aldehyde stabilizing solution
US5998483A (en) * 1991-09-20 1999-12-07 Camiener; Gerald W. Glyoxal-containing preservative compositions

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4242291A (en) * 1979-05-09 1980-12-30 Battelle Development Corporation Collagen reconstitution
US4361552A (en) * 1980-09-26 1982-11-30 Board Of Regents, The University Of Texas System Wound dressing
US5077218A (en) * 1988-05-05 1991-12-31 Oregon Lions Sight And Hearing Foundation Inc. Eye transport apparatus
US5328821A (en) * 1991-12-12 1994-07-12 Robyn Fisher Cold and cryo-preservation methods for human tissue slices
US10969210B2 (en) 2018-04-25 2021-04-06 Pro-Tracker Ip Holding, Llc System and method for adjusting the trajectory of an arrow

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